A secreted cysteine protease (CP) fraction from Trichomonas vaginalis is shown here to induce apoptosis in human vaginal epithelial cells (HVEC) and is analyzed by mass spectrometry. The trichomonad parasite T. vaginalis causes one of the most common non-viral sexually transmitted infection in humans, trichomoniasis. The parasite as well as a secreted cysteine protease (CP) fraction, isolated by affinity chromatography followed by Bio-Gel P-60 column chromatography, are shown to induce HVEC apoptosis, as demonstrated by the Cell Death Detection ELISA(PLUS) assay and annexin V-fluorescein isothiocyanate flow cytometry analyses. Initiation of apoptosis is correlated with protease activity because the specific CP inhibitor E-64 inhibits both activities. SDS-PAGE analysis of the CP fraction reveals triplet bands around 30 kDa, and matrix-assisted laser desorption ionization time-of-flight MS indicates two closely associated peaks of molecular mass 23.6 and 23.8 kDa. Mass spectral peptide sequencing of the proteolytically digested CPs results in matches to previously reported cDNA clones, CP2, CP3, and CP4 (Mallinson, D. J., Lockwood, B. C., Coombs, G. H., and North, M. J. (1994) Microbiology 140, 2725-2735), as well as another sequence with high homology to CP4 (www.tigr.org). These last two species are the most abundant components of the CP fraction. The present results, suggesting that CP-induced programmed cell death may be involved in the pathogenesis of T. vaginalis infection in vivo, may have important implications for therapeutic intervention.

We identified a novel Drosophila protein of approximately 400 kDa, hemolectin (d-Hml), secreted from haemocyte-derived Kc167 cells. Its 11.7 kbp cDNA contains an open reading frame of 3843 amino acid residues, with conserved domains in von Willebrand factor (VWF), coagulation factor V/VIII and complement factors. The d-hml gene is located on the third chromosome (position 70C1-5) and consists of 26 exons. The major part of d-Hml consists of well-known motifs with the organization: CP1-EG1-CP2-EG2-CP3-VD1-VD2-VD'-VD3-VC1-VD"-VD"'-FC1-FC2-VC2-LA1-VD4-VD5-VC3-VB1-VB2-VC4-VC5-CK1 (CP, complement-control protein domain; EG, epidermal-growth-factor-like domain; VB, VC, VD, VWF type B-, C- and D-like domains; VD', VD", VD"', truncated C-terminal VDs; FC, coagulation factor V/VIII type C domain; LA, low-density-lipoprotein-receptor class A domain; CK, cysteine knot domain). The organization of VD1-VD2-VD'-VD3, essential for VWF to be processed by furin, to bind to coagulation factor VIII and to form interchain disulphide linkages, is conserved. The 400 kDa form of d-Hml was sensitive to acidic cleavage near the boundary between VD2 and VD', where the cleavage site of pro-VWF is located. Agarose-gel electrophoresis of metabolically radiolabelled d-Hml suggested that it is secreted from Kc167 cells mainly as dimers. Resembling VWF, 7.9% (305 residues) of cysteine residues on the d-Hml sequence had well-conserved positions in each motif. Coinciding with the development of phagocytic haemocytes, d-hml transcript was detected in late embryos and larvae. Its low-level expression in adult flies was induced by injury at any position on the body.

Vasopressin is a peptide hormone that regulates renal water excretion in part through its actions on the collecting duct. The regulation occurs in part via control of transcription of genes coding for the water channels aquaporin-2 (Aqp2) and aquaporin-3 (Aqp3). To identify transcription factors expressed in collecting duct cells, we have carried out LC-MS/MS-based proteomic profiling of nuclei isolated from native rat inner medullary collecting ducts (IMCDs). To maximize the number of proteins identified, we matched spectra to rat amino acid sequences using three different search algorithms (SEQUEST, InsPecT, and OMSSA). All searches were coupled to target-decoy methodology to limit false-discovery identifications to 2% of the total for single-peptide identifications. In addition, we developed a computational tool (ProMatch) to identify and eliminate ambiguous identifications. With this approach, we identified >3,500 proteins, including 154 proteins classified as "transcription factor" proteins (Panther Classification System). Among these, are members of CREB, ETS, RXR, NFAT, HOX, GATA, EBOX, EGR, MYT1, KLF, and CP2 families, which were found to have evolutionarily conserved putative binding sites in the 5'-flanking region or first intron of the Aqp2 gene, as well as members of EBOX, NR2, GRE, MAZ, KLF, and SP1 families corresponding to conserved sites in the 5'-flanking region of the Aqp3 gene. In addition, several novel phosphorylation sites in nuclear proteins were identified using the neutral loss-scanning LC-MS(3) technique. The newly identified proteins have been incorporated into the IMCD Proteome Database (http://dir.nhlbi.nih.gov/papers/lkem/imcd/).

The synthesis of juvenile hormone (JH) is an attractive target for control of insect pests and vectors of disease, but the minute size of the corpora allata (CA), the glands that synthesize JH, has made it difficult to identify important biosynthetic enzymes by classical biochemical approaches. Here, we report identification and characterization of an insect farnesol dehydrogenase (AaSDR-1) that oxidizes farnesol into farnesal, a precursor of JH, in the CA. AaSDR-1 was isolated as an EST in a library of the corpora allata-corpora cardiaca of the mosquito Aedes aegypti. The 245-amino acid protein presents the typical short-chain dehydrogenase (SDR) Rossmann-fold motif for nucleotide binding. This feature, together with other conserved sequence motifs, place AaSDR-1 into the "classical" NADP(+)-dependent cP2 SDR subfamily. The gene is part of a group of highly conserved paralogs that cluster together in the mosquito genome; similar clusters of orthologs were found in other insect species. AaSDR-1 acts as a homodimer and efficiently oxidizes C(10) to C(15) isoprenoid and aliphatic alcohols, showing the highest affinity for the conversion of farnesol into farnesal. Farnesol dehydrogenase activity was not detected in the CA of newly emerged mosquitoes but significant activity was detected 24 h later. Real time PCR experiments revealed that AaSDR-1 mRNA levels were very low in the inactive CA of the newly emerged female, but increased >30-fold 24 h later during the peak of JH synthesis. These results suggest that oxidation of farnesol might be a rate-limiting step in JH III synthesis in adult mosquitoes.

Several lines of evidence suggest that some of the neurotoxicity in Alzheimer's disease (AD) is attributed to proteolytic fragments of amyloid precursor protein (APP) and beta-amyloid (Abeta) may not be the sole active component involved in the pathogenesis of AD. The potential effects of other cleavage products of APP need to be explored. The CTFs, carboxy-terminal fragments of APP, have been found in AD patients' brain and reported to exhibit much higher neurotoxicity in a variety of preparations than Abeta. Furthermore CTFs are known to impair calcium homeostasis and learning and memory through blocking LTP, triggering a strong inflammatory reaction through MAPKs- and NF-kappaB-dependent astrocytosis and iNOS induction. Recently, it was reported that CTF translocated into the nucleus, binding with Fe65 and CP2, and in turn, affected transcription of genes including glycogen synthase kinase-3beta, which results in the induction of tau-rich neurofibrillary tangles and subsequently cell death. Spatial memory of transgenic (Tg) mice overexpressing CT100 was significantly impaired and CTFs were detected in the neurons as well as in plaques of the Tg mice and double Tg mice carrying CT100 and mutant tau. In this review, we summarize observations indicating that both CTF and Abeta may participate in the neuronal degeneration in the progress of AD by differential mechanisms.

The mammalian transcription factor LSF (CP2/LBP-1c) binds cellular promoters modulated by cell growth signals. We demonstrate here that LSF-DNA-binding activity is strikingly regulated by induction of cell growth in human peripheral T lymphocytes. Within 15 min of mitogenic stimulation of these cells, the level of LSF-DNA-binding activity increased by a factor of five. The level of LSF protein in the nucleus remained constant throughout this interval. However, a rapid decrease in the electrophoretic mobility of LSF, attributable to phosphorylation, correlated with the increase in DNA-binding activity. pp44 (ERK1) phosphorylated LSF in vitro on the same residue that was phosphorylated in vivo, specifically at amino acid position 291, as indicated by mutant analysis. As direct verification of the causal relationship between phosphorylation and DNA-binding activity, treatment in vitro of LSF with phosphatase both increased the electrophoretic mobility of the protein and decreased LSF-DNA-binding activity. This modulation of LSF-DNA-binding activity as T cells progress from a resting to a replicating state reveals that LSF activity is regulated during cell growth and suggests that LSF regulates growth-responsive promoters.

Duct epithelial structure is an essential feature of many internal organs, including exocrine glands and the kidney. The ducts not only mediate fluid transfer but also help to maintain homeostasis. For instance, fluids and solutes are resorbed from or secreted into the primary fluid flowing through the lumen of the ducts in the exocrine glands and kidneys. The molecular mechanism underlying the functional maturation of these ducts remains largely unknown. Here, we show that a grainyhead-related transcription factor, CP2-like 1 (CP2L1), is required for the maturation of the ducts of the salivary gland and kidney. In the mouse, Cp2l1 is specifically expressed in the developing ducts of a number of exocrine glands, including the salivary gland, as well as in those of the kidney. In Cp2l1-deficient mice, the expression of genes directly involved in functional maturation of the ducts was specifically reduced in both the salivary gland and kidney, indicating that Cp2l1 is required for the differentiation of duct cells. Furthermore, the composition of saliva and urine was abnormal in these mice. These results indicate that Cp2l1 expression is required for normal duct development in both the salivary gland and kidney.

In source leaves of resistant tobacco, oxidative burst and subsequent formation of hypersensitive lesions after infection with Phytophthora nicotianae was prevented by inhibition of glucose-6-phosphate dehydrogenase (G6PDH) or NADPH oxidases. This observation indicated that plant defense could benefit from improved NADPH availability due to increased G6PDH activity in the cytosol. A plastidic isoform of the G6PDH-encoding gene, G6PD, displaying high NADPH tolerance was engineered for cytosolic expression (cP2), and introduced into a susceptible cultivar. After infection, transgenic (previously susceptible) lines overexpressing cP2 showed early oxidative bursts, callose deposition, and changes in metabolic parameters. These responses resulted in timely formation of hypersensitive lesions similar to resistant plants, although their extent varied considerably between different transgenic lines. Additional RNAi suppression of endogenous cytosolic G6PD isoforms resulted in highly uniform defense responses and also enhanced drought tolerance and flowering. Cytosolic G6PDH seems to be a crucial factor for the outcome of plant defense responses; thus, representing an important target for modulation of stress resistance. Because isoenzyme replacement of G6PDH in the cytosol was beneficial under various kinds of cues, we propose this strategy as a tool to enhance stress tolerance in general.

Congenital erythropoietic porphyria, an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of uroporphyrinogen III synthase. Extensive mutation analyses of 40 unrelated patients only identified approximately 90% of mutant alleles. Sequencing the recently discovered erythroid-specific promoter in six patients with a single undefined allele identified four novel mutations clustered in a 20-bp region: (a) a -70T to C transition in a putative GATA-1 consensus binding element, (b) a -76G to A transition, (c) a -86C to A transversion in three unrelated patients, and (d) a -90C to A transversion in a putative CP2 binding motif. Also, a -224T to C polymorphism was present in approximately 4% of 200 unrelated Caucasian alleles. We inserted these mutant sequences into luciferase reporter constructs. When transfected into K562 erythroid cells, these constructs yielded 3 +/- 1, 54 +/- 3, 43 +/- 6, and 8 +/- 1%, respectively, of the reporter activity conferred by the wild-type promoter. Electrophoretic mobility shift assays indicated that the -70C mutation altered GATA1 binding, whereas the adjacent -76A mutation did not. Similarly, the -90C mutation altered CP2 binding, whereas the -86A mutation did not. Thus, these four pathogenic erythroid promoter mutations impaired erythroid-specific transcription, caused CEP, and identified functionally important GATA1 and CP2 transcriptional binding elements for erythroid-specific heme biosynthesis.

The functions of the Alzheimer's beta-amyloid precursor protein (APP) and of its complex with the adaptor protein Fe65 are still unknown. We have demonstrated that Fe65 is also a nuclear protein and APP functions as an extranuclear anchor, thus preventing Fe65 nuclear translocation. According to this finding, it was also demonstrated that Fe65 could play a role in the regulation of transcription. In the present paper we show that the overexpression of Fe65 prevents G(1) ->> S cell cycle progression of serum-stimulated fibroblasts and that the contemporary overexpression of APP abolishes this effect of Fe65. The overexpression of Fe65 completely abolishes the activation of a key S phase gene, the thymidylate synthase (TS) gene, driven by the transcription factor LSF/CP2/LBP1 (LSF). This phenomenon is observed only in experimental conditions leading to the accumulation of Fe65 in the nucleus. Similarly, the two other members of the Fe65 protein family, Fe65L1 and Fe65L2, have been found to translocate into the nucleus and to prevent the activation of the TS gene promoter induced by LSF. Two results support the hypothesis that the inhibitory effect of Fe65 on cell cycle progression in fibroblasts is the result of the inhibition of TS gene expression: (i) Fe65 overexpression, but not Fe65 and APP co-expression, prevents the accumulation of endogenous TS upon the exposure of cells to serum, and (ii) thymidine addition to the culture medium completely overcomes the growth arrest caused by Fe65. In neuronal PC12 cells, the overexpression of Fe65 or of Fe65L1 and Fe65L2 blocks cell cycle, as observed in fibroblasts, but thymidine supplementation to culture medium does not revert this block, thus suggesting that Fe65 proteins induce in neuronal cells a gene expression program different from that activated in fibroblasts.

The proteins responsible for erythroid-specific footprints extending to -180 on the mouse alpha-globin gene were identified, enriched, and characterized from extracts of murine erythroleukemia (MEL) cells. Three proteins accounted for most aspects of the footprints. The binding sites of two proteins, termed alpha-CP1 and alpha-CP2, overlapped in the CCAAT box. Further characterization of these two CCAAT binding proteins showed that neither interacted with the adenovirus origin of replication, a strong CCAAT transcription factor-nuclear factor 1 binding site. A third protein, termed alpha-IRP, interacted with two sequences that formed an inverted repeat (IR) between the CCAAT and TATAA boxes. Interestingly, the binding domain of one of the CCAAT factors, alpha-CP1, overlapped one alpha-IRP binding site. alpha-CP1 thus overlapped the binding domains of both alpha-CP2 and alpha-IRP. The IRs included GC-rich sequences reminiscent of SP1-binding sites. Indeed, alpha-IRP bound as well to the alpha-promoter as it did to SP1 sites in the simian virus 40 early promoter. These results suggest that alpha-IRP may be related to the transcription factor Sp1. We determined the level of each alpha-globin-binding activity before and after induced erythroid differentiation of MEL cells. We found that differentiation caused alpha-CP1 activity to drop three- to fivefold, while alpha-IRP activity decreased slightly and alpha-CP2 activity increased two- to threefold.

A distinctive tract of all the forms of Alzheimer's disease is the extracellular deposition of a 40-42/43 amino acid-long peptide derived from the so-called beta-amyloid precursor protein (APP). This is a membrane protein of unknown function, whose short cytosolic domain has been recently demonstrated to interact with several proteins. One of these proteins, named Fe65, has the characteristics of an adaptor protein; in fact, it possesses three protein-protein interaction domains: a WW domain and two PID/PTB domains. The interaction with APP requires the most C-terminal PID/PTB domain, whereas the WW domain is responsible for the interaction with various proteins, one of which was demonstrated to be the mammalian homolog of the Drosophila enabled protein (Mena), which in turn interacts with the cytoskeleton. The second PID/PTB domain of Fe65 binds to the CP2/LSF/LBP1 protein, which is an already known transcription factor. The other proteins interacting with the cytosolic domain of APP are the G(o) heterotrimeric protein, APP-BP1 and X11. The latter interacts with APP through a PID/PTB domain and possesses two other protein-protein interaction domains. The small size of the APP cytodomain and the overlapping of its regions involved in the binding of Fe65 and X11 suggest the existence of competitive mechanisms regulating the binding of the various ligands to this cytosolic domain. In this short review the possible functional roles of this complex protein network and its involvement in the generation of Alzheimer's phenotype are discussed.

A library of Streptococcus gordonii DL1-Challis DNA was constructed in lambda gt11. Phage plaques were screened for production of antigens that reacted with antiserum to S. gordonii cell surface proteins. A recombinant phage denoted lambda gt11-cp2 was isolated that carried 1.85 kb of S. gordonii DNA and that expressed an antigen with a molecular mass of 29 kDa in Escherichia coli. Antibodies that reacted with the expression product were affinity purified and were shown to react with a single polypeptide antigen with a molecular mass of 76 kDa in S. gordonii DL1-Challis. A segment (0.85 kb) of the cloned DNA within the transcription unit was ligated into a nonreplicative plasmid carrying an erythromycin resistance determinant and transformed into S. gordonii DL1-Challis. The plasmid integrated onto the chromosome, and expression of the 76-kDa polypeptide antigen was abolished. The gene inactivation had no obvious effect on bacterial growth or on a number of phenotypic properties, including hydrophobicity and adherence. However, it abolished serum-induced cell aggregation, mutant cells had reduced aggregation titers in saliva and in colostrum immunoglobulin A, and it also reduced coaggregation with some Actinomyces species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of cell envelope proteins from wild-type and mutant strains showed that as well as lacking the surface-exposed 76-kDa polypeptide, mutant cell envelopes were deficient in several other polypeptides, including those that bound to immunoglobulin A. Expression of the gene encoding the 76-kDa polypeptide in S. gordonii appeared to be critical for functional conformation of the cell surface.

Two mutations that affect larval cuticle protein gene expression in the 2/3 variant Drosophila melanogaster strain were investigated. We demonstrate that this strain synthesizes an electrophoretic variant, fast 2 (CPf2), of wild-type cuticle protein 2(CP2). It also lacks detectable amounts of cuticle protein 3 (CP3). The other major cuticle proteins are still present. Protein and DNA sequence analyses indicate that point mutations cause two amino acid substitutions that change the electrophoretic mobility of CPf2 relative to that of CP2. The mutation abolishing the expression of CP3 was found to be a 7.3-kilobase DNA insertion located within the T-A-T-A box region of this gene, at -31 base pairs from the mRNA start site. This DNA insertion, called H.M.S. Beagle, belongs to a conserved family of repeated DNA elements that have characteristics similar to those of previously characterized Drosophila transposable elements. H.M.S. Beagle elements are repeated approximately 50 times in the haploid genome and exhibit restriction fragment-length polymorphisms around points of insertion between Canton S, Oregon R, and 2/3 Drosophila strains. Sequence analysis indicates that H.M.S. Beagle contains 266-base-pair direct repeats at its termini and is flanked by a duplication of 4 base pairs of target DNA sequence, T-A-T-A, in the CP3 gene insertion. Thus, insertion of a transposable element into the putative promoter region of the CP3 gene is evidently responsible for inactivating CP3 gene expression.

A gene bank of Campylobacter pylori DNA in Escherichia coli was constructed by cloning Sau3A-cleaved DNA fragments into the bacteriophage vector lambda EMBL3. The expression of C. pylori antigens was determined by screening the gene library with adsorbed C. pylori whole-cell rabbit antisera. One recombinant clone which reacted positively (lambda CP2) was studied further. Immunoblot analysis with lambda CP2 showed a polypeptide band of 66 kilodaltons (kDa) reacting antigenically with the adsorbed antiserum. Extraction of DNA from lambda CP2 and digestion with SalI revealed a DNA insert of 17 kilobases (kb). Subcloning with SalI and the E. coli vector pUC18 showed that the DNA also encoded a 31-kDa antigen. The cloned antigens were shown by immunoblotting to have the same molecular weight in E. coli as in C. pylori and to be present in all C. pylori strains. Antiserum was raised against the cloned polypeptides and found to react only with C. pylori when analyzed by dot blotting and indirect immunofluorescence. The cloned antigens were determined to be expressed from the pUC18 lac promoter. The DNA encoding these antigens was radiolabeled with 32P and found to hybridize only to C. pylori strains. Immunoblotting with affinity-purified polyclonal antibody to the urease enzyme of C. pylori revealed that the cloned antigens may be part of the urease enzyme.

Glycosylphosphatidylinositol (GPI) anchoring of membrane proteins occurs through two distinct steps, namely the assembly of a precursor glycolipid and its subsequent transfer onto newly synthesized proteins. To analyze the structure of the yeast precursor glycolipid we made use of the pmi40 mutant that incorporates very high amounts of [3H]mannose. Two very polar [3H]mannose-labeled glycolipids named CP1 and CP2 qualified as GPI precursor lipids since their carbohydrate head group, Man alpha 1,2(X->>PO4-->6)Man alpha 1,2Man alpha 1,6Man alpha-GlcN-inositol (with X most likely being ethanolamine) comprises the core structure which is common to all GPI anchors described so far. CP1 predominates in cells grown at 24 degrees C whereas CP2 is induced by stress conditions. The apparent structural identity of the head groups suggests that CP1 and CP2 contain different lipid moieties. The lipid moieties of both CP1 and CP2 can be removed by mild alkaline hydrolysis although the protein-bound GPI anchors made by the pmi40 cells under identical labeling conditions contain mild base resistant ceramides. These findings imply that the ceramide moiety found on the majority of yeast GPI anchored proteins is added through a lipid remodeling step that occurs after the addition of the GPI precursor glycolipids to proteins.

The apical organelles in apicomplexan parasites are characteristic secretory vesicles containing complex mixtures of molecules. While apical organelle discharge has been demonstrated to be involved in the cellular invasion of some apicomplexan parasites, including Toxoplasma gondii and Plasmodium spp., the mechanisms of apical organelle discharge by Cryptosporidium parvum sporozoites and its role in host cell invasion are unclear. Here we show that the discharge of C. parvum apical organelles occurs in a temperature-dependent fashion. The inhibition of parasite actin and tubulin polymerization by cytochalasin D and colchicines, respectively, inhibited parasite apical organelle discharge. Chelation of the parasite's intracellular calcium also inhibited apical organelle discharge, and this process was partially reversed by raising the intracellular calcium concentration by use of the ionophore A23187. The inhibition of parasite cytoskeleton polymerization by cytochalasin D and colchicine and the depletion of intracellular calcium also decreased the gliding motility of C. parvum sporozoites. Importantly, the inhibition of apical organelle discharge by C. parvum sporozoites blocked parasite invasion of, but not attachment to, host cells (i.e., cultured human cholangiocytes). Moreover, the translocation of a parasite protein, CP2, to the host cell membrane at the region of the host cell-parasite interface was detected; an antibody to CP2 decreased the C. parvum invasion of cholangiocytes. These data demonstrate that the discharge of C. parvum sporozoite apical organelle contents occurs and that it is temperature, intracellular calcium, and cytoskeleton dependent and required for host cell invasion, confirming that apical organelles play a central role in C. parvum entry into host cells.

Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins. After solubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p is found in 430-650-kDa protein complexes. These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p, and Gpi16p (YHR188c). Gpi16p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, and it has a single C-terminal transmembrane domain and a small C-terminal, cytosolic extension with an ER retrieval motif. Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins. Gpi8p and Gpi16p are unstable if either of them is removed by depletion. Similarly, when Gpi8p is overexpressed, it largely remains outside the 430-650-kDa transamidase complex and is unstable. Overexpression of Gpi8p cannot compensate for the lack of Gpi16p. Homologues of Gpi16p are found in all eucaryotes. The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively. When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact.

We recently cloned human and murine cDNAs that encode CP2, a transcription factor that interacts with the murine alpha-globin promoter. In this report, we exploited our ability to express CP2 in bacteria and eukaryotic cells to further investigate factor activities in vitro and in vivo. CP2 expressed in bacteria was significantly enriched and used in a series of DNase I footprinting and electrophoretic gel shift assays. The results suggest that CP2 binds a hyphenated recognition sequence motif that spans one DNA helix turn. In addition, the enriched bacterial protein activated transcription of alpha-globin promoter templates approximately 3- to 4-fold in vitro. We then tested the effect of elevating CP2 levels 2.5- to 5.5-fold in vivo using both transient and stable transformation assays. When a reporter construct comprised of the intact murine alpha-globin promoter driving the bacterial chloramphenicol acetyltransferase (CAT) gene was introduced into these overexpressing cells, we observed a 3- to 6-fold increase in CAT activity when compared to cells expressing normal levels of CP2. These results define the CP2 factor binding site in more detail and help characterize the activities of the factor in vivo.

We have previously purified four factors (alpha-IRP, alpha-CP1, alpha-CP2, and NF-E1) that interact with the promoter of the alpha-globin gene. One of these (NF-E1) is a tissue-restricted factor that has recently been cloned. The binding sites of these factors identify DNA sequence elements that might mediate the tissue-specific and inducible transcription of the alpha-globin gene. This possibility was tested in a series of in vitro transcription experiments. An examination of 5' truncated templates and synthetic promoters constituted from individual factor-binding sites apposed to the alpha-TATAA box showed that the binding elements of three factors (alpha-CP1, alpha-IRP, and NF-E1) mediate four- to sixfold activation of transcription in vitro. In contrast, one element (alpha-CP2) stimulated transcription less than twofold. The 5- to 10-fold stimulation of these latter templates upon addition of a DNA sequence affinity-purified factor suggests that alpha-CP2 is functionally limiting in nuclear extracts. Additional experiments further tested the effect of supplementing extracts with factors purified from erythroid cell nuclear extracts or, in the case of NF-E1, enriched from a bacterial cDNA expression system. Each factor tested stimulated transcription in vitro in a binding-site-dependent manner. Our results provide a comprehensive functional view of the murine alpha-globin promoter and suggest possible mechanisms for activation of alpha-globin gene transcription during induced differentiation of murine erythroleukemia cells.

The RNA-binding protein human antigen R (HuR) has been implicated in apoptosis in multiple ways. Several studies have shown that in response to a variety of stresses HuR promotes the expression of proapoptotic mRNAs, whereas others reported its regulatory effect on antiapoptotic messages. We recently showed that in response to severe stress, HuR is cleaved to generate two cleavage products (CPs), HuR-CP1 (24 kDa) and HuR-CP2 (8 kDa), by which it promotes apoptotic cell death. Here, we show that this cleavage event is dependent on protein kinase RNA (PKR). Surprisingly, although in response to the apoptotic inducer staurosporine PKR itself is not phosphorylated, PKR triggers the cleavage of HuR via its downstream effector FADD that in turn activates the caspase-8/caspase-3 pathway. This effect, however, does not require the phosphorylation of the eukaryotic translation initiation factor 2alpha. Additionally, we observed that these HuR-CPs are sufficient to trigger cell death in the absence of activation of the PKR pathway. Therefore, our results support a model whereby in response to lethal stress, PKR, without being phosphorylated, activates the FADD/caspase-8/caspase-3 pathway to trigger HuR cleavage, and the HuR-CPs are then capable of promoting apoptosis.

To investigate the synergism or cooperative interaction between transcription elements, we have designed and constructed a series of synthetic polymerase II promoters with different combinations of elements. These include three different CCAAT boxes, which correspond to the binding sites for CP1, CP2, and NFI, a GC box, a CACCC box, and an ATF/CREB-binding site. The synthetic promoters containing these elements in proximal positions were linked to a test gene (CAT). Tandem repeats of AP1- and AP2-binding sites, the simian virus 40 enhancer, and DNA-binding sites for GAL-estrogen receptor were cloned downstream of the test gene. The strength of these promoters was then tested in transient-expression assays in HeLa TK- cells. In the context of the adenovirus major late promoter TATA box, the promoters containing only certain combinations of elements are active in this assay. Some elements appear to cooperate nearly universally, but others exhibit strong selectivity. These results indicate strongly selective synergistic interactions between elements and suggest that levels of promoter strength may be determined by the extent of compatibility between factors bound to proximal and enhancer sites.

Although the varepsilon4 allele of the apolipoprotein E gene appears as an important biological marker for Alzheimer's disease (AD) susceptibility, other genetic determinants are clearly implicated in the AD process. Here, we propose that a genetic variation in the transcriptional factor LBP-1c/CP2/LSF gene, located close to the LRP locus, is a genetic susceptibility factor for AD. We report an association between a non-coding polymorphism (G->>A) in the 3'-untranslated region of this gene and sporadic AD in French and British populations and a similar trend in a North American population. The combined analysis of these three independent populations provides evidence of a protective effect of the A allele (OR = 0.58, 95% CI 0.44-0.75). We describe a potential biologically relevant role for the A allele whereby it reduces binding to nuclear protein(s). The absence of the A allele was associated with a lower LBP-1c/CP2/LSF gene expression in lymphocytes from AD cases compared with controls. Our data suggest that polymorphic variation in the implication of the LBP-1c/CP2/LSF gene may be important for the pathogenesis of AD, particularly since LBP-1c/CP2/LSF interacts with proteins such as GSKbeta, Fe65 and certain factors involved in the inflammatory response.

The discovery of small molecule inhibitors of cytotoxicity induced by amyloid-beta (Abeta) oligomers, either applied extracellularly or accumulated intraneuronally, is an important goal of drug development for Alzheimer's disease (AD), but has been limited by the lack of efficient screening methods. Here we describe our approach using two cell-based methods. The first method takes advantage of the unique ability of extracellularly applied Abeta oligomers to rapidly induce the exocytosis of formazan formed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We employed a short protocol to quantify this toxicity, and quickly identified two novel inhibitors, code-named CP2 and A5, from two compound libraries. A second independent screen of the same libraries using our previously published MC65 protection assay, which identifies inhibitors of toxicity related to intracellular Abeta oligomers, also selected the same two leads, suggesting that both assays select for the same anti-Abeta oligomer properties displayed by these compounds. We further demonstrated that A5 attenuated the progressive aggregation of existing Abeta oligomers, reduced the level of intracellular Abeta oligomers, and prevented the Abeta oligomer-induced death of primary cortical neurons, effects similar to those demonstrated by CP2. Our results suggest that, when combined, the two methods would generate fewer false results and give a high likelihood of identifying leads that show promises in ameliorating Abeta oligomer-induced toxicities within both intraneuronal and extracellular sites. Both assays are simple, suitable for rapid screening of a large number of medicinal libraries, and amenable for automation.